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Scalable Geometrically Designed Protein Cages Assembled via Genetically Encoded Split Inteins

50 Pages Posted: 21 Nov 2018 Sneak Peek Status: Under Review

See all articles by James N. Wright

James N. Wright

Queen Mary University of London - School of Biological and Chemical Sciences

Wan Ling Wong

Queen Mary University of London - School of Biological and Chemical Sciences

Joseph A. Harvey

Queen Mary University of London - School of Biological and Chemical Sciences

James A. Garnett

Queen Mary University of London - School of Biological and Chemical Sciences

Laura S. Itzhaki

University of Cambridge - Department of Pharmacology

Ewan Main

Queen Mary University of London - School of Biological and Chemical Sciences

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Abstract

Engineering proteins to assembly into user-defined structures is key in their development for biotechnological applications. However, designing generic rather than bespoke solutions is a challenging task. Here, we describe an expandable recombinant assembly system that produces scalable protein cages via split intein-mediated native chemical ligation. Three types of construct are used: a pair of oligomeric mirror-image “half-cages” and an extendable “linker". All constructs are composed of modular protein domains chosen to fulfil the required geometries, fused to two orthogonal pairs of split-intein halves to drive assembly when mixed. This combination enables both one-pot construction of two-component cages and stepwise assembly of larger three-component scalable cages. To illustrate the system’s versatility, tetrahedral half-cages and linker constructs comprising consensus-designed repeat proteins were ligated in one-pot and stepwise reactions. Under mild conditions rapid high yielding ligations were obtained, from which discrete proteins cages were easily purified and shown to form the desired trigonal bipyramidal structures.

Keywords: directed protein assembly, native chemical ligation, protein conjugation, split-intein ligation, protein design, protein semi-synthesis

Suggested Citation

Wright, James N. and Wong, Wan Ling and Harvey, Joseph A. and Garnett, James A. and Itzhaki, Laura S. and Main, Ewan, Scalable Geometrically Designed Protein Cages Assembled via Genetically Encoded Split Inteins (November 21, 2018). Available at SSRN: https://ssrn.com/abstract=3288532 or http://dx.doi.org/10.2139/ssrn.3288532
This is a paper under consideration at Cell Press and has not been peer-reviewed.

James N. Wright

Queen Mary University of London - School of Biological and Chemical Sciences

London
United Kingdom

Wan Ling Wong

Queen Mary University of London - School of Biological and Chemical Sciences

London
United Kingdom

Joseph A. Harvey

Queen Mary University of London - School of Biological and Chemical Sciences

London
United Kingdom

James A. Garnett

Queen Mary University of London - School of Biological and Chemical Sciences

London
United Kingdom

Laura S. Itzhaki

University of Cambridge - Department of Pharmacology

Trinity Ln
Cambridge, CB2 1TN
United Kingdom

Ewan Main (Contact Author)

Queen Mary University of London - School of Biological and Chemical Sciences ( email )

London
United Kingdom

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